Methods and compositions for treatment of angiogenic diseases

ABSTRACT

The subject invention pertain to methods of treating Kaposi&#39;s Sarcoma and ocular neovascularization using rPF4 and angiogenesis-inhibiting fragments of rPF4.

This is a continuation of application Ser. No. 07/822,378, filed Jan.16, 1992, now abandoned; which is a continuation-in-part of applicationSer. No. 07/600,472, filed Oct. 19, 1990, now U.S. Pat. No. 5,284,827;which is a division of application Ser. No. 07/451,021, filed Dec. 27,1989, now U.S. Pat. No. 5,086,164; which is a continuation-in-part ofapplication Ser. No. 07/295,955, now abandoned.

BACKGROUND OF THE INVENTION

The field of the invention is angiogenesis.

Angiogenesis, the development of new capillary blood vessels, is animportant process in the developing fetus and growing human. However, inhealthy adults, angiogenesis occurs significantly only during woundhealing and in the menstrual cycle.

It is now widely recognized that much of the angiogenic activityoccurring in adults is pathological in nature. For example,proliferation of vascular endothelial cells and formation of newcapillaries is essential for growth of solid tumors beyond a few cubicmillimeters in volume (Folkman et al. 1983! Ciba Found. Symp.100:132-149). We now understand that developing tumors secrete growthfactors which stimulate neighboring endothelial cells to divide andmigrate toward the tumor.

In addition to growth of solid tumors, other conditions involvingangiogenic dysfunctions include diabetic retinopathy, retrolentalfibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune andnon-immune inflammation (including rheumatoid arthritis), capillaryproliferation within atherosclerotic plaques, hemangiomas, and Kaposi'sSarcoma have also recently been recognized as diseases possessingcharacteristics of dysregulated endothelial cell division and capillarygrowth. These conditions along with growth of solid tumors arecollectively referred to as "angiogenic diseases" (Folkman, J., and M.Klagsbrun 1987! Science 235:442-447).

In addition to angiogenic diseases, there are other conditions whereendothelial cell proliferation is pathological or, at least, unwanted.For example, endometriosis is characterized by the abnormalproliferation and positioning of certain endothelial cells whichnormally line the inner wall of the uterus. Control of the angiogenicprocess could help to prevent or alleviate endometriosis. Also,prevention of endothelial cell growth in the uterus could be a means ofbirth control.

Endothelial cell growth is associated with wound healing. This growth isundesirable during extended surgical proceedings and where excessivescar formation may occur. Therefore, a means of controlling endothelialcell proliferation would help prevent or reduce unwanted scar formation.

The mechanism of angiogenesis and endothelial cell proliferation has notbeen completely characterized. It has been established that mast cellsaccumulate at a tumor site before new capillary growth occurs; however,mast cells alone cannot initiate angiogenesis. Heparin, a mast cellproduct, has been shown to significantly stimulate the capillaryendothelial cell migration which is necessary for angiogenesis (Folkman,J. 1984! Angiogenesis: Initiation and Modulation. In Cancer Invasion andMetastasis: Biologicand Therapeutic Aspects. G. L. Nicolson and L.Milas, eds. Raven Press, New York, pp. 201-208).

Several substances are known to have the capability of inhibitingendothelial cell growth in vitro. One of the most extensively studiedinhibitors of endothelial cell growth is protamine, which is a proteinfound only in sperm. Protamine has been shown to inhibit tumorangiogenesis and subsequent tumor growth (Taylor, S. and J. Folkman1982! Nature 297:307-312). Protamine's anti-angiogenesis activity hasbeen attributed to its well-known capacity to bind heparin (Taylor andFolkman 1982!, supra). Clinical experiments with protamine have not beenpursued because of the toxicity associated with protamine injection.Protamine, which is usually isolated from salmon sperm, is known to beantigenic in humans, and anaphylactic reactions to this protein havebeen observed with secondary exposures.

At least two other compounds have been studied in regard to theirheparin-binding activity: platelet factor 4 (PF4) and major basicprotein. Major basic protein has demonstrated heparin-binding activitybut is of little practical utility because of its high toxicity.

Platelet factor 4 is a well-known protein which has been completelysequenced (Deuel, T. F., R. M. Senior, D. Chang, G. L. Griffin, R. L.Heinrikson, and E. T. Kaiser 1981! Proc. Natl. Acad. Sci. USA78:4585-4587). It is a 70-residue secretable platelet protein with amolecular weight of approximately 7.8 Kd which is released duringplatelet aggregation. Although there is evidence of heparin bindingactivity and some indications of anti-angiogenesis activity (Folkman1984!, supra), PF4 has never been shown to have clinical utility.

A compound which has been described as "oncostatin A," and which appearsto be the same, or similar to, native PF4, has been implicated asaffecting the growth of tumors (U.S. Pat. Nos. 4,645,828 and 4,737,580;both issued to Twardzik et al.). However, the effects reported in thesepatents pertain to slowly growing human cancer cells in immunodeficientmice. The results of these experiments cannot be reliably extrapolatedto predict the effect of rapidly growing tumors which are native to thehost animal. Furthermore, the experiments reported in these patents inno way predict or disclose any angiostatic properties.

Various peptides from PF4 have been purified and their propertiesstudied. None has been shown to have any role in the inhibition ofangiogenesis. It is known that the C-13 peptide of PF4 is chemotacticfor neutrophils and monocytes (Osterman, D. G., G. L. Griffin, R. M.Senior, E. T. Kaiser, and T. H. Deuel 1982! Biochem. and Biophys. Res.Comm. 107(1):130-135). It is significant to note that the infiltrationof monocytes would be expected to stimulate the proliferation andmigration of local endothelial cells by the secretion of angiogenicfactors. Thus, peptides of PF4 could be expected to stimulate, ratherthan inhibit, angiogenesis.

In addition to angiostatic properties, PF4 possesses characteristicstructural features of the pro-inflammatory proteins interleukin-8 andβ-thromboglobulin and has been shown to be chemotactic for neutrophilsand monocytes in vivo (Wolpe and Cerami 1989! the FASEB Journal,3:2565-2573). This similarity of the structure and activities of PF4 towell characterized pro-inflammatory proteins along with the ubiquitousaggregation of platelets at sites of inflammation suggest that PF4 maybe an endogenous mediator of inflammation. Thus, it is anticipated thatswelling could accompany the administration of PF4 in vivo.

There is a significant and very long-standing need to locate aneffective and non-toxic inhibitor of angiogenesis and endothelial cellproliferation. Angiogenesis plays a major role in the initiation andprogression of widespread catastrophic illnesses, including cancer. Aneffective, non-toxic agent which can be administered-locally and/orsystemically to treat these illnesses would be highly advantageous andhas long eluded identification.

The following table may be helpful in identifying the amino acids of thesubject invention:

    ______________________________________                                                        Three-letter                                                                            One-letter                                          Amino acid      symbol    symbol                                              ______________________________________                                        Alanine         Ala       A                                                   Arginine        Arg       R                                                   Asparagine      Asn       N                                                   Aspartic acid   Asp       D                                                   Asn and/or Asp  Asx       B                                                   Cysteine        Cys       C                                                   Glutamine       Gln       Q                                                   Glutamic acid   Glu       E                                                   Gln and/or Glu  Glx       Z                                                   Glycine         Gly       G                                                   Histidine       His       H                                                   Isoleucine      Ile       I                                                   Leucine         Leu       L                                                   Lysine          Lys       K                                                   Methionine      Met       M                                                   Phenylalanine   Phe       F                                                   Proline         Pro       P                                                   Serine          Ser       S                                                   Threonine       Thr       T                                                   Tryptophan      Trp       W                                                   Tryrosine       Tyr       Y                                                   Valine          Val       V                                                   ______________________________________                                    

When used herein, the term conservative amino acid substitution meansthe substitution of an amino acid with another amino acid that isbiologically compatible with the first amino acid.

SUMMARY OF THE INVENTION

This invention concerns the discovery that PF4 has clinical utility inthe treatment of diseases which involve angiogenesis and endothelialcell proliferation. Furthermore, PF4 fragments are demonstrated to beinhibitors of angiogenesis. The ability to inhibit angiogenesis has beenfound in synthetic peptides corresponding to sequences in PF4 as smallas the carboxyterminal 13 amino acids.

The invention features treatment of angiogenic diseases with acombination of PF4 and an anti-inflammatory agent. Anti-inflammatoryagents help to alleviate unwanted swelling, pain, or tissue damage whichcould accompany the administration of pro-inflammatory compounds.

The invention also features methods for the treatment of tumors thatcontain malignant endothelial cells with PF4 either alone or incombination with an anti-inflammatory agent. In particular, theinvention features methods for the treatment of brain tumors with PF4.

DETAILED DESCRIPTION

The drawings are first described.

The Drawings

FIG. 1 shows DNA and amino acid sequence of native rPF4.

FIG. 2 shows the inhibition of angiogenesis resulting from the treatmentof rPF4 and various related peptides.

FIG. 3 depicts the inhibition of endothelial cell proliferation by rPF4.

FIG. 4 depicts the alpha-helical configurations of rPF4 and rPF4-241.

FIG. 5 compares the inhibition of angiogenesis resulting from treatmentwith rPF4 and rPF4-241.

FIG. 6 compares the inhibition of human umbilical vein endothelial cellproliferation resulting from treatment with rPF4 or rPF4-241.

FIG. 7 shows the ability of rPF4 and rPF4-241 to inhibit tumor growth.

FIG. 8 shows footpad swelling in mice as a function of time afterinjection with either rPF4, rPF4 and indomethacin, or a buffer solution.

FIG. 9 shows quantification of inflammatory cell infiltrate aftertreatment with rPF4 or rPF4 with indomethacin.

FIG. 10 shows tumor growth after administration of rPF4 alone,indomethacin alone, buffer alone, or rPF4 and indomethacin.

The subject invention pertains to in vivo inhibition of angiogenesis byrPF4 and certain analogs and peptide fragments of PF4. These analogs andpeptide fragments of PF4 can be used to treat angiogenic diseases. Asused in this application, the term "angiogenic disease" refers to growthof-solid tumors, and other conditions involving angiogenic dysfunctionsincluding diabetic retinopathy, retrolental fibroplasia, ocularneovascularization, corneal neovascularization induced by trauma,infection or irritation, neovascular glaucoma, macular degeneration,psoriasis, angiofibromas, immune and non-immune inflammation (includingarthritis and rheumatoid arthritis), capillary proliferation withinatherosclerotic plaques, hemangiomas, and Kaposi's sarcoma. The subjectinvention also concerns the use of rPF4 and PF4 fragments for treatmentof diseases of dysregulated endothelial cell proliferation.

The types of solid tumors that can be treated by rPF4, PF4, or analogsand fragments thereof, include all types of lung tumors, including smallcell lung carcinoma, tumors of the breast, colon/rectum, prostate, headand neck, stomach, bladder, kidney, pancreas, liver, ovary and uterus;sarcomas; melanoma and other metastatic skin cancers; nonmetastatic skincancers (e.g., Kaposi's sarcoma, basal cell carcinoma); and, mostpreferably tumors of the brain.

The types of tumors that can treated using the compositions and methodsof the invention include tumors that are not surgically accessibleand/or are resistant to chemotherapy and radiation therapy. Advancedinvasive malignancy, with or without surgery, can be treated using thecompositions and methods of the invention. The invention can be used asan adjuvant therapy following surgical resection and can be used totreat known metastatic disease and nonmetastatic cancer.

Treatment of the tumors and diseases described above can be eithersystemic, regional, or local (intralesional), depending upon the typeand severity of the disease as well as the accessibility of the diseasesite. Systemic treatment includes intravenous bolus injections andinfusions, subcutaneous injections, implants; refillable reservoirs andsustained release depots and intramuscular injections. As will beexplained in more detail below, the inventors have discovered thateffective anti-tumor dosages useful in systemic administration must bevery high; these unusually high dosages, which are necessary for maximumefficacy, can be administered with acceptably low attendant toxicity.Regional treatment includes intraarterial for the treatment of primaryliver tumors and liver metastases, and for the treatment of kidney,brain and pancreatic tumors. Regional intraperitoneal treatment can beused for the treatment of tumors of the ovary. Local treatment can beused for tumors of the brain, uterus, bladder, head and neck, forKaposi's sarcoma and other nonmetastatic skin cancers, for metastaticskin cancer once dissemination precludes further surgical excision, andfor colon and rectal cancer.

Brain tumors are generally treated with initial surgical excision ifpossible, followed by intensive chemotherapy and radiation therapy.Aggressive brain tumors (high grade astrocytoma, glioblastomamultiforme) ultimately produce mortality by reoccurrence near theiroriginal site due to incomplete surgical removal, rather than throughmetastatic dissemination. Methods to specifically focus postsurgicaltreatment on the site of the initial lesion are therefore desirable todeliver effective therapy and reduce damage to healthy tissues.

The subject invention in part takes advantage of the ability of rPF4 toinhibit capillary formation in vivo as well as embryonicneovascularization. Full length recombinant PF4 also inhibits growthfactor-dependent human endothelial cell proliferation in vitro.

Significantly, it was determined that the angiogenesis-inhibitingactivity of PF4 was retained by synthetic peptides corresponding tosequences of PF4 as small as 13 amino acids in length. In particular, itwas found that a synthetic peptide of 13 amino acids corresponding tothe carboxyl terminal portion of PF4 (C-13) displayed potent angiostaticactivity.

The finding that PF4 directly inhibits growth of pure cultures ofendothelial cells indicates that, advantageously, its effects are notmediated by some other cell type.

The activity of the C-13 peptide is especially surprising in light ofits inability to affect the anticoagulant activity of heparin. The useof the C-13 peptide offers several advantages over whole rPF4 such asreduced dosage (weight basis), reduced likelihood of antigenicity, andgreater likelihood of effectiveness in novel dosage forms.

The C-13 peptide of PF4 also retains the ability to prevent Con-Ainduced immunosuppression in mice, an activity which is unaffected byheparin and probably independent of the ability of the peptide toinhibit angiogenesis.

It is well understood that angiogenesis is required for solid tumors togrow beyond a few cubic millimeters. Thus for the treatment of solidtumors, use of rPF4, or a fragment thereof, to inhibit tumor growth byinhibiting angiogenesis presents a novel and highly advantageous meansof therapy, although efficacy of PF4 in some of the therapies describedherein is not entirely explained by inhibition of angiogenesis; forexample, we have discovered that in specific types of cancers thatcontain malignant endothelial cells, such as Kaposi's sarcoma, PF4 wasinhibitory.

The fact that the C-13 peptide inhibits angiogenesis without affectingthe anticoagulant activity of heparin demonstrates that this smallpeptide would also have the benefit of not interfering with concurrentanticoagulant therapy. Additionally, small peptides are generally lessantigenic than larger proteins, and, thus, the PF4 fragments can be usedadvantageously for oral and transdermal administration. These types ofdelivery are particularly useful in the treatment of gastrointestinalcapillary proliferation (e.g., Kaposi's Sarcoma) and skin lesions,respectively. Intralesional, as well as systemic, administration of PF4fragments are also appropriate for treatment of these conditions.

Analogs of PF4 were created which lack heparin binding activity butretain ability to inhibit angiogenesis. One such analog, known asrPF4-241, was created by cassette mutagenesis of a synthetic PF4 genewhereby the DNA sequence encoding the four lysine residues near thecarboxy terminus of PF4 were converted to a sequence encoding twoGln-Glu couplets. If rPF4-241 is administered intralesionally, it can beapplied such that the dosage is between about 1 μg/lesion and about 4mg/lesion. For systemic administration, the dosage of rPF4-241 can bebetween 0.5 mg/kg of body weight and about 100 mg/kg of body weight.Similar and higher dosages can be used for the administration of nativesequence rPF4 as well as peptide fragments. For example, dosages of rPF4and fragments thereof may be twice that of rPF4-241 or higher.

As discussed above, PF4 has been shown to be chemotactic for neutrophilsand monocytes in vitro, suggesting that it may mediate an inflammatoryresponse. To assess whether these observations have in vivo relevance,the ability of PF4 to induce acute and chronic dermal inflammation inthe mouse was tested. When injected into the murine dermis, recombinanthuman PF4 (rPF4) induces acute inflammation within two hours, whichpeaks at about 12 to 18 hours and which resolves by about 36 hours.Injection of an equivalent amount of cytochrome c, buffer alone, or anamino terminal PF4 peptide failed to elicit a significant inflammatoryresponse, however, the carboxy terminal PF4 peptide waspro-inflammatory. The inflammatory infiltrate induced by both rPF4 andthe 41 amino acid COOH terminal peptide was composed of neutrophils andto a lesser degree mononuclear cells. Although relatively highconcentrations of rPF4 are required to elicit an inflammatory response,these concentrations may be locally obtainable during plateletaggregation or at sites of administration of rPF4 or related compounds.

Advantageously, it was found that the rPF4 pro-inflammatory effect wassignificantly suppressed by systemic administration of ananti-inflammatory agent without reducing the angiostatic activity.

Materials and Methods

Chicken Chorioallantoic Membrane (CAM) Assay.

Fertile eggs were incubated in a stationary position for 3 days at 37°C. and 70-80% relative humidity. During this time, the embryo rose tothe upper surface of the egg contents. At the beginning of the 4th day,the eggs were cracked without inversion and carefully deposited intosterile plastic petri dishes such that the embryo remained on the uppersurface. The shell-free eggs were incubated for an additional 72 hoursat 37° C., under an atmosphere containing 2.5-3.5% CO₂ after which thegrowing embryos developed a recognizable CAM. Discs, made by mixing testsamples with 1% (w/v) methylcellulose, were dried and placed on the CAMbetween major veins and approximately 0.5 cm from the embryo. Followinganother 48 hour incubation at 37° C. (2.5-3.5% CO₂), the samples werescored for their ability to inhibit angiogenesis. Inhibition appears asan avascular zone surrounding the implant and can often include elbowsformed by veins avoiding the disc and a reduced number of capillaries inthe region of the implant.

Endothelial Cell Proliferation Assay.

Human umbilical vein endothelial cells (HUVEC) were cultured in Medium199 (Gibco) containing 10% (v/v) fetal bovine serum (FBS), 150 mcg/mlendothelial cell growth supplement (ECGS) and 5 units/ml heparin at 37°C. and 4-5% CO₂. Every 3-4 days, the cultures were harvested by trypsintreatment, diluted, replated, and grown to confluence. Prior to thestart of an experiment, the cells were centrifuged and resuspended inheparin-free media and incubated with the test substance,(PF4) for 3days under standard culture conditions. At the end of the incubationperiod, the cells were removed by trypsin treatment and counted with aParticle Data Elzone 180 Cell Counter. Statistical significance betweenmeans was determined by a standard Student t-test for unpaired data.

Inhibition of DNA synthesis was measured by plating the cells asdescribed, then incubating with the test substance for 24 hours. ³H-Thymidine (1 μCi/well) was added for an additional 6 hours and theplates were frozen at -70° C. Following 2 freeze/thaw cycles, the cellswere aspirated onto a fiber filter, washed with distilled water, fixedwith MeOH, and counted for incorporation of radioactivity into DNA.

In vivo Tumor Growth Assay.

Normal C57BL/6J female mice (6-8 weeks old) were inoculatedsubcutaneously with 5×10⁵ log phase cells of a B16-F10 melanoma tumorline. This protocol led to progressive tumor growth resulting in large(300 mm³) necrotic tumors after approximately 10 days, followed by deathof untreated animals usually within three weeks of tumor inoculation.

To test the efficacy of rPF4 in preventing in vivo tumor growth andangiogenesis, tumor bearing animals were injected daily, directly intothe nascent tumor, with either rPF4 or with buffer lacking rPF4,beginning one day after tumor inoculation. Tumor volume was measured atregular intervals with digital calipers by laboratory personneluninformed of the specific treatment received by each subject animal.

Footpad Assay.

0.05 ml of PBS containing a test substance was injected intradermallyinto the right hind footpad of each mouse. An identical amount ofdiluent, not containing the test substance, was injected into the lefthind footpad. At various time points, footpad thicknesses were measuredwith a spring loaded engineer's micrometer (Fowler Co., Biggswald,England).

At various time points, mice were sacrificed and footpad tissue wasprepared for light microscopy. This tissue was used to quantifyinfiltrating cell types. Biopsy specimens were fixed in 10% bufferedformalin for at least 48 hours and then prepared using standardtechniques of paraffin embedding and staining with hematoxylin andeosin. Using an ocular grid, four cellular areas of dermis in eachspecimen were examined in a coded fashion at 1000×magnification andinflammatory cells were quantified. Differences between groups wereassessed by Student's t test or analysis of variance, where appropriate.

rPF4 Production.

Recombinant PF4 was produced in E. coli as an N-terminal fusion proteincontaining a unique methionine residue immediately preceding the PF4portion. More specifically, expression plasmid pPF4-211 was constructedby cloning a synthetic gene encoding native sequence PF4 (FIG. 1) (Ponczet al. 1987! Blood 69:219) into the multiple restriction site region ofplasmid pREV2.2 (deposited Jul. 30, 1986; accession # NRRL B-18091).Codon usage in the synthetic gene was optimized for expression in E.coli, and synthetic DNA linkers were included on each end to facilitatethe directional insertion of the PF4 gene into the vector. Therestriction sites HindIII and SmaI were chosen for insertion intopREV2.2. The resulting construct, pPF4-211, expressed a fusion proteincontaining 34 amino acids of E. coli β-glucuronidase (BG) separated fromthe PF4 sequence by a unique methionine residue.

Cells expressing the fusion protein were subjected to lysozyme (1 mg/gcells), DNase I (500 units/100 g cells) and bead mill treatments. Thelysis pellet containing the fusion protein was treated with CNBr (10g/100 g cells) in 70% formic acid to cleave the fusion protein at themethionine between the BG and PF4 portions. Following evaporation of theCNBr/formic acid, the recombinant protein was extracted with 200 ml of50 mM Tris-Cl, pH 7.6, 5 mM EDTA, and 10 mM DTT per 100 g of cellstarting material. Native sequence rPF4-211 was purified by binding theprotein to heparin agarose, removing contaminating proteins with 0.6MNaCl, and eluting with 1.2M NaCl. The resulting material was dialyzedinto 20 mM sodium acetate, pH 4.0, and analyzed on a 15% SDS-PA gelstained with Coomassie Brilliant Blue. Minor contaminants could beremoved using C₄ reverse phase high pressure liquid chromatography(HPLC) to prepare the protein for in vivo use.

Production of rPF4-241 and other PF4 analogs.

A synthetic gene encoding the mutant designated rPF4-241 was constructedby changing the codons for the four lysine residues near the C-terminusof PF4 to sequences encoding two Gln-Glu couplets (CAA GAA) by cassettemutagenesis between the BbeI and SmaI sites. Linkers were included atthe ends of the synthetic gene, and the gene was inserted into pREV2.2as described above. Genes encoding other PF4 mutants or analogs wereprepared in a similar manner.

The mutant proteins (e.g., rPF4-241) were cleaved and extracted asdescribed above. The extracts were then purified using DEAE-Sepharosechromatography, and eluted with a gradient of 0-1M NaCl. The PF4proteins generally eluted at approximately 0.5M NaCl and were dialyzedinto 20 mM phosphate buffer, pH 7.5. The samples were further purifiedby reverse: phase HPLC.

PF4 peptides.

Peptides were prepared by standard solid phase synthesis procedures,cleaved from the solid support and deblocked, and purified by reversephase HPLC.

Reagents.

Recombinant human IL-1 (rIL-1) was purchased from Genzyme Corporation(Cambridge, Mass). Cytochrome c and E. coli endotoxin were purchasedfrom Sigma Chemical Co. (St. Louis, Mo.). Slow release indomethacinpellets were purchased from Innovative Research (Toledo, Ohio).

Mice.

C57Bl/6J, A/J and C3H/HeJ female mice, 6-8 weeks old, were purchasedfrom the Jackson Laboratory (Bar Harbor, Me.).

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1

Chicken eggs, prepared as described above, were treated with discscontaining several concentrations of recombinant PF4 or peptides derivedfrom the sequence of PF4. rPF4 and C-terminal peptides as small as 13amino acids inhibited angiogenesis on the CAM (FIG. 2). In each case,the inhibition was dose-dependent and the response approximatelyequivalent (molar basis) for the inhibitors containing the C-terminalregion of PF4. An N-terminal peptide of PF4 (N-29) did not inhibitangiogenesis even at the highest concentration tested, suggesting thatall of the anti-angiogenic activity of PF4 is probably associated withthe C-terminal portion of the molecule. Since the C-terminus of PF4 isrich in lysine, polylysine was tested in this assay system and found notto cause inhibition at 6.5 nmol dosages.

EXAMPLE 2

The lysine rich region of PF4 (residues 61-66) is also the domainassociated with the binding of heparin by PF4. Heparin is known to playa role in modulating angiogenesis, which can also be affected byprotamine, another well characterized heparin-binding protein. To assessthe ability of PF4-based synthetic peptides to bind heparin, we assayedthe activity of coagulation-cascade enzymes which are inhibited byheparin. The Factor Xa assay used here has previously been described inDenton et al. (1983) Biochem. J. 209:455-460. Protamine and plateletfactor 4 are able to prevent the heparin inhibition of thrombin andFactor Xa at approximately equimolar concentrations. The 41 amino acidC-terminal peptide of PF4 (C-41) prevented heparin inhibition lesseffectively, but the C-13 peptide was unable to prevent the inhibitionof thrombin even at concentrations ten times that of an effective levelof rPF4. This unexpected finding suggests that the C-13 peptide inhibitsangiogenesis by some method other than heparin binding.

EXAMPLE 3

Many angiostatic agents act by direct inhibition of endothelial cellproliferation. Endothelial cell division and growth is tightlycontrolled and strictly dependent on the presence of growth factors. Weevaluated the ability of rPF4 having the wild type sequence (rPF4-211)and related peptides to inhibit growth factor-stimulated humanendothelial cell proliferation in vitro. As shown in FIG. 3, rPF4significantly inhibited endothelial cell growth in a dose-dependentfashion at a concentration as low as 1.3 μM. Inhibition was complete at3.2 μM in the heparin-deficient medium employed here.

EXAMPLE 4

To assess the importance of the heparin binding activity of PF4 in theinhibition of endothelial cell proliferation, cells were incubated inmedia containing or lacking 5 units/ml heparin. The presence of heparinstimulated proliferation of these cells during the three day incubationof this experiment. rPF4 significantly inhibited, both control (100%)and heparin stimulated (45%) endothelial cell growth (Table 1).

                  TABLE 1                                                         ______________________________________                                        Attenuation of rPF4 inhibition of                                             endothelial cell growth by heparin.                                                     rPF4           %                                                    Addition   --          50 mcg/ml Inhibition.sup.a                             ______________________________________                                        --         14.4 ± 2.5                                                                              .sup.b 6.0 ± 0.6                                                                    ˜100                                   5 u/ml heparin                                                                           18.9 ± 1.2                                                                             .sup.b 14.0 ± 0.4                                                                     45                                          ______________________________________                                         .sup.a Based on seeding of 8 × 10.sup.4 cells/well                      .sup.b Significantly different from appropriate control (p < 0.005)      

EXAMPLE 5

Construction of rPF4-241

A mutant of PF4 was created by converting the four lysine residues atthe carboxy terminus of PF4 to two Gln-Glu couplets as disclosed above.This protein apparently retains the alpha-helical secondary structure(FIG. 4) for this region of the molecule with the concurrent loss ofheparin binding activity.

The protein was reactive with polyclonal antibodies to native PF4 andwas determined to possess the appropriate modifications by amino acidanalysis. Significantly, the purified mutant protein lackedheparin-binding activity in the Factor Xa inhibition assay.

The substitutions described here can be made with the peptide fragmentsas well as with the full length PF4 molecule. For example, C-13-241 hasthe following sequence:

Pro-Leu-Tyr-Gln-Glu-Ile-Ile-Gln-Glu-Leu-Leu-Glu-Ser

EXAMPLE 6

Inhibition of Angiogenesis by rPF4-241

Purified rPF4-241 was tested for its ability to inhibit capillary growthin the chicken chorioallantoic membrane (CAM) assay. Even at the lowestconcentrations tested (1.25 nmol/disc) rPF4-241 extensively inhibitedangiogenesis in the CAM system (FIG. 5). This inhibition was even moreeffective than that caused by equal concentrations of native rPF4 assuggested by larger avascular zones on the membrane. The inhibitoryeffect of rPF4-241 was not reversed by heparin.

EXAMPLE 7

Inhibition of Human Endothelial Cell Proliferation by rPF4-241

In a test of inhibition of human umbilical vein endothelial cellproliferation by native rPF4 and mutant rPF4-241, both were shown to beeffective at inhibiting the proliferation of these cells. The results ofthis test are shown in FIG. 6.

These results are remarkable in that previous theories of PF4 inhibitionof angiogenesis assumed that the PF4 effects were due to heparinbinding. We have designed a protein, retaining most of the structuralfeatures of native PF4 but lacking detectable heparin binding activity,which may be more active than native PF4 in inhibiting angiogenesis invivo and endothelial cell proliferation in vitro. Additionally, themutant we have designed would not be expected to interfere with heparinanticoagulant therapy.

EXAMPLE 8

Inhibition of In Vivo Tumor Growth

The efficacy of rPF4-211 or rPF4-241 in preventing tumor growth andangiogenesis was tested. The inhibition of in vivo tumor growth wasassayed after injection of either rPF4-211 (in 20 mM NaOAc, pH 4.0) orrPF4-241 (in 50 mM sodium phosphate, pH 6.5, 50 mM NaCl) directly intothe nascent tumor, as described in the materials and methods sectionabove. Within seven days of tumor inoculation, animals injected withbuffer possessed obvious three dimensional tumors, whilerPF4-211-treated animals were essentially tumor-free (FIG. 7). Continuedtreatment with rPF4 completely suppressed tumor growth under theseconditions where control animal tumors became necrotic and large as seenpreviously with untreated mice. The same effect was observed whenrPF4-241 was used as the inhibitory agent.

This finding supports the proposition that rPF4, as an inhibitor ofangiogenesis, will possess clinical usefulness in the management ofmalignant melanoma and other cancers. Progressive growth of tumorsrequires new blood vessel formation which, if inhibited, may not onlyrestrict tumor growth, but stimulate regression of existing vessels, aswell as enhance other responses to malignant invasion.

The finding that rPF4 inhibition of in vivo tumor growth was apparentwithin three days of the initial inoculation (of rPF4) indicates thatrPF4 acts to modulate tumor growth by local mechanisms rather than byimmunomodulation which would require a longer time course. Additionally,rPF4 did not directly inhibit tumor cell growth in vitro. It appears,therefore, that rPF4 modulated the host's angiogenic response to thegrowing tumor.

EXAMPLE 9

It has been shown that proteins of identified structure and function maybe constructed by changing the amino acid sequence if such changes donot significantly alter the protein secondary structure (Kaiser, E. T.,and F. J. Kezdy 1984! Science 223:249-255). The subject inventionincludes other mutants or fragments of the PF4 sequences depicted hereinwhich lack affinity for heparin and exhibit substantially the same orhigher angiostatic activity. A preferred region for modification is thelysine rich region near the carboxy terminus corresponding to theheparin binding domain (residues 60-70). As a general rule, amino acids60 through 70 cannot be eliminated. Also, as a general rule, it isnecessary to have at least one charged residue between positions 60 and70. Maintenance of an amphipathic α-helix in this region does not seemto be necessary, however, an amphipathic structure may be preferable.Thus, the subject invention includes mutants of the amino acid sequencesdepicted herein which do not alter the protein secondary structure, orif the structure is altered, the biological activity is retained. Inparticular it should be understood that conservative substitutions ofamino acids may be made. For example, amino acids may be placed in thefollowing classes: basic, hydrophobic, acidic, polar, and amide.Substitutions whereby an amino acid of one class is replaced withanother amino acid of the same type fall within the scope of the subjectinvention so long as the substitution does not materially alter thebiological activity of the compound. Table 2 provides a listing ofexamples of amino acids belonging to each class.

                  TABLE 2                                                         ______________________________________                                        Class of Amino Acid                                                                             Example of Amino Acids                                      ______________________________________                                        Basic             K, R, H                                                     Hydrophobic       A, L, I, V, P, F, W, Y, M                                   Acidic            E, D                                                        Polar             S, T, N, Q, C                                               Amide             Q, N                                                        ______________________________________                                    

In some instances, non-conservative substitutions can also be made. Forexample, a lysine residue near the C-terminus of PF4 may be replacedwith any of the following amino acids: E, Q, D, N, M, A, L, and I. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the rPF4 or the rPF4 fragment.

We have conducted experiments whereby amino acid substitutions have beenmade, and the resulting rPF4 mutants have been tested for biologicalactivity. Various mutants which have been constructed are shown in Table3.

                  TABLE 3                                                         ______________________________________                                        Designation                                                                              Sequence                                                           ______________________________________                                                                    60                                                        70                                                                    P L Y K K I I K K L L L E S                                                   P L Y231    PF4 AA 1-57!                                                      P L Y Q E I I Q E L L E S                                                     P L Y Q Q I I Q Q L L E S                                                     P L Y K K Q E K K Q E E S                                                     P L Y Q I E I Q L E L E S                                                     P L Y N D I I N D L L E S                                                     P L Y G E I I G E L L E S                                                     ______________________________________                                    

Results from experiments testing the biological activity of thesepeptides are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                      CAM  HUVEC                                                      ______________________________________                                        rPF4-211        +      +                                                      rPF4-231        +/-    -                                                      rPF4-241        ++     +                                                      rPF4-302        +/-    -                                                      rPF4-303        +      NA                                                     rPF4-307        ++     ++                                                     rPF4-308        +      NA                                                     rPF4-315        +      NA                                                     ______________________________________                                         NA = Not available                                                       

The results shown in Table 4 clearly demonstrate that it is possible tomake rPF4 mutant which retain the biological activity of rPF4 withrespect to inhibition of cell growth in the CAM assay and the HUVECassay. Two of these peptides (rPF4-241 and rPF4-307) exhibited enhancedactivity in these assays. The mutants described here are amino acidsequences which are largely homologous with wild type rPF4 (rPF4-211),but which have certain amino acid substitutions. These substitutionswere made between amino acids 60 and 70.

Although most of the resulting compounds still exhibit biologicalactivity in the CAM and HUVEC assays, they do not bind heparin.rPF4-302, which does not exhibit significant activity in either the CAMor the HUVEC assay, has no charged amino acid residues between residues60 and 70. rPF4-231, which also does not exhibit significant biologicalactivity, terminates at amino acid number 60. If a person skilled in theart wished to investigate the biological activity of other rPF4 mutants,it would now be a straightforward procedure to make the desiredmutations and test the resulting peptides for activity. Using theteachings of this document, the researcher could prepare and readilytest peptides which could be expected to have the desired properties.For example, the amino acid substitutions just described for the fulllength rPF4 molecule can also be made with the C-13 and C-41 fragmentswhich are described above.

EXAMPLE 10

Inflammatory Properties of rPF4 and Related Compounds

The inflammatory properties of rPF4 and related compounds were assessedusing the footpad assay as described above. At 8 hours, local injectionof 25 μg of rPF4-211 into the murine dermis resulted in a briskinflammatory response as measured by footpad swelling (FIG. 8) andquantification of inflammatory cell infiltrate (FIG. 9). At higher dosesthe tissue edema does not increase further and may even drop offslightly. It has been found that relatively high local concentrations ofPF4 are required to exert a pro-inflammatory effect. Although a briskinflammatory response occurs with 25 μg of PF4 injected into the murinedermis, at 0.25 μg, the inflammatory response is minimal. The timecourse of rPF4 induced acute inflammation is broad and resolves by about36 hours (FIG. 8).

The time course of rPF4 induced inflammation shows a rapid increase frombaseline and peaks at between 6 and 12 hours and almost completelyresolves by 36 hours.

EXAMPLE 11

Effects of Anti-Inflammatory Agent with rPF4

For each mouse, 0.05 mg, slow release indomethacin pellets (InnovativeResearch, Toledo, Ohio) were implanted subcutaneously under light etheranesthesia 48 hours prior to an experiment. These pellets continuouslyrelease their contents over 14 days.

Systemic treatment of animals with indomethacin significantly blunts therPF4 pro-inflammatory response (FIG. 8). The area under the curve offootpad swelling in the rPF4 plus indomethacin treated mice is 45.7% ofthe area under the curve of the rPF4 alone treated mice. Theinflammatory cell infiltrate is also partially abrogated withindomethacin treatment. The results of these experiments are summarizedin Table 5.

                  TABLE 5                                                         ______________________________________                                                       Pro-inflammatory response                                      Treatment        Swelling Infiltrating                                        ______________________________________                                        rPF4             ++       ++                                                  C-41             ++       ++                                                  N-29             -        -                                                   rPF4-241         +        +                                                   rPF4/indomethacin                                                                              +/-      +/-                                                 ______________________________________                                    

Thus indomethacin can be used to decrease the swelling which couldaccompany the administration of PF4 or PF4-related substances. Othernon-steroidal anti-inflammatory agents could also be used. Theanti-inflammatory agents useful in the combinations and methods of thisinvention include steroidal and non-steroidal anti-inflammatory agents.The non-steroidal anti-inflammatory agents include, but are not limitedto, acetyl salicylic acid (aspirin), methyl salicylate, sodiumsalicylate, phenylbutazone, oxyphenbutazone, apazone, indomethacin,sulindac, tolmetin, mefenamic acid, ibuprofen, naproxen, fenoprofen,flurbiprofen, ketoprofen, and other compounds. Other anti-inflammatoryagents useful in the combinations and methods of this invention arelipocortins derived from natural sources or lipocortins andlipocortin-like polypeptides produced by recombinant techniques (seeU.S. patent applications Ser. Nos. 690,146; 712,376; 765,877 and772,892; Wallner, B. et al. 1986! Nature 320:77-81) and uromodulin(Muchmore, A. V., and J. M. Decker 1985! Science 229:479-481), orcyclosporin and its derivatives. Steroidal anti-inflammatory agentswhich could be used according to the subject invention include, but arenot limited to hydrocortisones.

EXAMPLE 12

Anti-Tumor Activity of rPF4 Combined with Indomethacin

Four groups of mice were used in this experiment. In two groups of mice,slow release indomethacin pellets (50 μg) were implanted surgicallyunder the skin of the left flank. The other two groups were not treatedwith indomethacin. Tumors were implanted subcutaneously in all fourgroups in the right flank.

As shown in FIG. 10, the addition of indomethacin to PF4 did notcompromise the antitumor activity of PF4. Implanted tumors grew rapidlyafter day 6 when the tumor was treated with either buffer alone orindomethacin alone. By contrast, the tumors grew very little, if at all,when treated with PF4 or a combination of PF4 and indomethacin.

From these results it is apparent that PF4 retains its antitumoractivity even when combined with the anti-inflammatory agentindomethacin.

EXAMPLE 13

Administration of PF4 and Anti-Inflammatory Agents

The combinations and methods of the present invention may allow theadministration of PF4, or related compounds, in higher doses in somecases than those tolerated in conventional treatment regimes based uponPF4 alone. Accordingly, the combinations and methods of this inventionadvantageously reduce or eliminate the inflammatory effects of high dosetreatments with PF4alone. Thus, the use of PF4 in combination with ananti-inflammatory agent may reduce the duration of treatment which wouldbe required by therapies based upon conventionally tolerated lowerdosages of PF4 alone.

The combinations and methods of this invention are useful in treatingany mammal, including humans. According to this invention, mammals aretreated with pharmaceutically effective amounts of the two activecomponents--PF4 and an anti-inflammatory agent--of the combinations ofthis invention for a period of time sufficient to inhibit angiogenesisor endothelial cell proliferation.

In accordance with this invention, pharmaceutically effective amounts ofan anti-inflammatory agent and the PF4 (or PF4-related compounds) areadministered sequentially or concurrently to the patient. The mosteffective mode of administration and dosage regimen of PF4 andanti-inflammatory agent will depend upon the type of disease to betreated, the severity and course of that disease, previous therapy, thepatient's health status, and response to PF4 and the judgment of thetreating physician. PF4 may be administered to the patient at one timeor over a series of treatments.

Preferably, the anti-inflammatory agent and the PF4 are administeredsequentially to the patient, with the anti-inflammatory agent beingadministered before, after, or both before and after treatment with PF4.Sequential administration involves treatment with the anti-inflammatoryagent at least on the same day (within 24 hours) of treatment with PF4and may involve continued treatment with the anti-inflammatory agent ondays that the PF4 is not administered. Conventional modes ofadministration and standard dosage regimens of anti-inflammatory agentsmay be used (see Gilman, A. G. et al. eds.! The Pharmacological Basis ofTherapeutics, pp. 697-713, 1482, 1489-91 1980!; Physicians DeskReference, 1986 Edition). For example, indomethacin may be administeredorally at a dosage of about 25-50 mg, three times a day. Higher dosesmay also be used. Alternatively, aspirin (about 1500-2000 mg/day),ibuprofen (about 1200-3200 mg/day), or conventional therapeutic doses ofother anti-inflammatory agents may be used. Dosages of anti-inflammatoryagents may be titrated to the individual patient.

According to one embodiment of this invention, the patient may receiveconcurrent treatments with the anti-inflammatory agent and PF4. Local,intralesional, or intravenous injection of PF4 is preferred (see Gilmanet al., supra at pp. 1290-91). The anti-inflammatory agent shouldpreferably be administered by subcutaneous injection, subcutaneousslow-release implant, or orally.

Alternatively, the patient may receive a composition comprising acombination of PF4 (or PF4-related compounds) and an anti-inflammatoryagent according to conventional modes of administration of agents whichexhibit anticancer, antitumor, or anti-inflammatory activity. Theseinclude, for example, parenteral, subcutaneous, intravenous, orintralesional routes of administration.

EXAMPLE 14

Dosages for Systemic Administration

It has been discovered that very high doses of PF4 are required when PF4is used systemically for treatment of tumors, and that these high dosesdo not have unacceptable high levels of toxicity. An experimentdemonstrating the need for, and tolerance of, high doses of PF4 wascarried out as follows.

Cells of a B16 murine melanoma cell line were injected intravenouslyinto a tail vein of a mouse. Thirty seconds later, rPF4 (in saline oracetate buffer) was injected into the same mouse via a different tailvein. After 21 days, the tumor burden of the test and control animalswas measured by counting, optically, lung metastases, and by weighingthe lungs of the mice. Referring to Table 6, PF4 produced adose-dependent effect, as measured by both parameters. Optimal resultswere observed when PF4 was administered systemically, when dosages inexcess of 5000 μg per kg of body weight; at these dosages, no observabletoxicity or other adverse effects were noted.

    ______________________________________                                                                      # of*                                                       Ave.*   Dose      Metastases                                                                           Lung*                                    Groups      Wt. (g) (mg/kg)   (#/Mouse)                                                                            Wt. (mg)                                 ______________________________________                                        Control     17.7                                                              0-                            136.2  443                                      (Buffer only)                                                                 0.375 mg rPF4                                                                             19.0    19.7      112.2  525                                      0.75 mg rPF4                                                                              18.7    40.1      66.0†                                                                         404                                      1.5 mg rPF4 17.8    84.2      54.7†                                                                         357                                      ______________________________________                                         *Average of six mice per group                                                †Statistically different from control group, p < 0.05.            

EXAMPLE 15

Treatment of Brain Tumors

Brain tumors will be treated with PF4 by itself, or in combination withradiation and or chemotherapy, as follows:

A. Local treatment following surgery

Following surgical removal of the primary tumor lesion, residual tumorgrowth is prevented by direct treatment with rPF4. Treatment methodsinclude direct injection of rPF4 into the lesion, implantation of slowrelease rPF4 formulations (biodegradable beads, etc.) or implantation ofa subcutaneous reservoir bearing a catheter for directing the deliveryof rPF4 to the specific location of the initial malignancy.

Formulations and dose will depend on the method of administration. Apreferred administration method is the direct injection or instillationof rPF4 by a subcutaneous reservoir/catheter system that is limited to0.05 to 5.0 ml total volume in a single dose. A single dose may containfrom 0.25 to 25 mg of rPF4 (0.003 to 0.4 mg/kg) and may be administeredas frequently as daily, wherein the patient receives one week of dailytreatment per month, or three times a week, wherein treatment isadministered continuously until significant patient improvement, ortreatment may be administered as infrequently as one time per month. Incertain situations it may be necessary to administer PF4 by continuousdirect administration over the course of several hours. Under suchconditions, the volume is increased to permit slower administration ofthe desired dose.

Tumors of the brain can also be treated by adopting methods widely usedin the treatment of gynecological and oral cancers, and are now appliedwith increasing frequency to brain and other cancers. Such methods,termed `brachytherapy`, involve the delivery of locally high doses ofradiotherapy (Cancer, Principles and Practice of oncology, ThirdEdition; DeVita, V. T. Jr., Hellman, S., Rosenberg, S. A. eds). Here,PF4 will be used in place of a radioactive agent. Implantable reservoirsfor intracranial infusion of therapeutic agents are widely available andinclude the Omaya reservoir and similar devices. Implantation of thereservoir can be done at the time of initial surgery or later in a minorsurgical procedure under local anaesthesia, by stereotactic methods.

B. Local treatment of unresectable lesions or recurrent lesions

Some brain tumors which are not resectable for technical and anatomicalreasons may be treated directly, without prior surgery, with therapeuticbenefit. Lesions which recur within one year of initial surgery are notgenerally appropriate for further surgical treatment and are treated byintratumoral methods. In this case, a preferred treatment isbrachytherapy, which is described above.

Brain tumors can be treated as described above on an out- or anin-patient basis, as the patient's health permits.

The potential therapeutic benefits of rPF4 to the patient are:

1) Restriction of tumor growth or indication of regression (as indicatedby lesion imaging and/or biopsy).

2) Improvement of symptoms associated with brain malignancy (incidenceof headache, memory function, personality disorders etc.) with orwithout observable effects on tumor progression.

3) Increased time to postsurgical recurrence.

4) Increased survival time.

EXAMPLE 16

Treatment of Kaposi's sarcoma, Psoriasis, Basal Cell Carcinoma and OtherSkin Disorders

Disorders of the skin can be treated intralesionally, whereinformulations and dose will depend on the method of administration and onthe size and severity of the lesion to be treated. Preferred methodsinclude intradermal and subcutaneous injection. Patients may be capableof self-administration. Preferred dosages are 0.05 to 5 mg rPF4 per dose(0.7 to 70 mg/kg) contained within a volume of 0.1 to 1 ml. Multipleinjections into large lesions may be possible, and several lesions onthe skin of a single patient may be treated at one time. The schedulefor administration can be the same as that described above for thetreatment of brain tumors. Formulations designed for sustained releasewill reduce the frequency of administration. Patients can be treated asout- or in- patients, as their health permits.

EXAMPLE 17

Systemic Treatment

Systemic treatment is essentially equivalent for all applications,including, but not limited to, small cell lung carcinoma, head and neckcancer, sarcoma, breast cancer, colon cancer, etc. PF4 can beadministered by direct intravenous injection, or preferably byintravenous infusion lasting from 0.5 to 4 hours per single treatment.Patients can be treated as in- or out-patients. Patients may also betreated using implantable subcutaneous portals, reservoirs, or pumps.Multiple intravenous or subcutaneous doses are possible, and in the caseof implantable methods for treatment, formulations designed forsustained release will be especially useful. Patients can be treated atdosages of 0.3 to 12 g of rPF4 per period; preferably with 4 to 180mg/kg in a volume of 60 ml to 2.5 liters per day.

A dosage is defined as a single dose administered as a bolus injectionor intravenous infusion; or the compound can be administered to thepatient as an intravenous infusion over a period of a day;alternatively, the compound can be administered in several bolusinjections interrupted by periods of time such that the dose isdelivered over the course of a 24 hour period. The most preferred methodof treatment is to administer the compound to the patient in oneinjection or infusion per day.

Patients may be treated daily on alternative weeks for six weeks, orpossibly for life. They may also be treated three times per weekcontinuously, or they may be treated daily for life.

EXAMPLE 18

Regional Treatment

Regional treatment is useful for treatment of cancers in specific organsin the patient, including, but not limited to primary liver cancer,brain and kidney cancer and liver metastases from colon/rectal cancer.Treatment can be accomplished by intraarterial infusion. A catheter canbe surgically or angiographically implanted to direct treatment to theaffected organ. A subcutaneous portal, connected to the catheter can beused for chronic treatment, or an implantable, refillable pump may alsobe employed. Patients can receive 0.05 to 1 g rPF4 (1 to 20 mg/kg) in avolume of 10 to 400 ml per single dose. The schedule for treatment isthe same as that described above for systemic treatment.

Compositions

The compositions used in these therapies may also be in a variety offorms. These include, for example, solid, semi-solid, and liquid dosageforms, such as tablets, pills, powders, liquid solutions or suspension,liposomes, suppositories, injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically acceptable carriers and adjuvants whichare known to those of skill in the art. Preferably, the compositions ofthe invention are in the form of a unit dose and will usually beadministered to the patient one or more times a day.

PF4, or related compounds, may be administered to the patient in anypharmaceutically acceptable dosage form, including intravenous,intramuscular, intralesional, or subcutaneous injection. An effectivedose may be in the range of from about 0.003 to about 200 mg/kg bodyweight, it being recognized that lower and higher doses may also beuseful. As discussed above, very high doses are preferred for systemicadministration. It should, of course, be understood that thecompositions and methods of this invention may be used in combinationwith other therapies.

Once improvement of the patient's condition has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, may be reduced, as a function ofthe symptoms, to a level at which the improved condition is retained.When the symptoms have been alleviated to the desired level, treatmentshould cease. Patients may, however, require intermittent treatment on along-term basis upon any recurrence of disease symptoms.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

I claim:
 1. A method for treating a patient with Kaposi's sarcoma, saidmethod comprising administering to said patient rPF4 or anangiogenesis-inhibiting fragment of rPF4, in a pharmaceuticallyacceptable carrier, wherein said rPF4 or said fragment or rPF4 isadministered either systemically or intralesionally at a dosagesufficient to deliver a sarcoma-inhibiting concentration of rPF4 to thesite of said Kaposi's sarcoma.
 2. The method, according to claim 1,wherein said rPF4 is administered either systemically at a dosage ofabout 0.3 g to about 12 g per dose or intralesionally at a dosage ofabout 0.05 mg to about 5 mg per dose.
 3. A method for treating a patientwith ocular neovascularization, said method comprising administering tosaid patient rPF4or an angiogenesis-inhibiting fragment of rPF4, in apharmaceutically acceptable carrier, wherein said rPF4 or said fragmentrPF 4 is administered either systemically or intraocularly at a dosagesufficient to deliver a neovascularization-inhibiting concentration ofrPF4 to said site of ocular neovascularization.
 4. The method, accordingto claim 3, wherein said rPF4 is administered either systemically at adosage of about 0.3 g to about 12 g per dose or intraocularly at adosage of about 0.05 mg to about 5 mg per dose.